Project Summary and Abstract Triple-negative breast cancer (TNBC) - an aggressive subtype of breast cancer that is associated with increased metastatic potential and poor patient survival - is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) and accounts for ~15- 20% of invasive breast cancers. TNBC represents an important clinical challenge because these cancers respond poorly to endocrine therapy or other available targeted agents; thus, chemotherapy is currently the backbone of standard therapy with a median survival of only ~13 months. Current FDA-approved nanoparticle- drug formulations of doxorubicin (Doxil) and paclitaxel (Abraxane) have been studied for the treatment of TNBC, however neither have been shown to significantly improve tumor control or patient survival. This is most likely due to (i) limited extravasation from the tumor vasculature, (ii) poor penetration within breast tumor tissue, (iii) inability to efficiently target tumor cell drug uptake within the tumor microenvironment, and (iv) development of drug resistance via expression of multidrug resistance (MDR) pumps such as P-glycoprotein. To address these therapeutic barriers, we have recently: (1) engineered relatively large polymeric nanoparticles (between 63 to 114 nm) that rapidly penetrate in breast tumor tissue with tumor-specific fibroblast growth factor-inducible 14 (Fn14)-targeting to further improve particle dispersion, drug distribution, and tumor-specific cellular uptake within the tumor microenvironment and (2) developed a novel high-throughput method for quantitative characterization of Fn14-specific and nonspecific binding (towards tumor ECM) of various nanoparticle formulations. Thus, the central hypothesis of this grant proposal is that by modulating the Fn14-specific equilibrium binding affinities (KD) and minimizing the nonspecific binding to tumor ECM, Fn14-targeted tumor penetrating nanoparticles will (1) provide well-dispersed, sustained delivery into the tumor and regions of the tumor tissue that contain TNBC cells and (2) specifically target to and efficiently traffic within Fn14-positive TNBC cells while sparing adjacent healthy tissues from toxic effects. This strategy is likely to result in significant improvements in efficacy and reduce toxicity in TNBC primary tumors and disseminated metastases, compared to free drugs and their clinical nanoparticle formulation counterparts, which will generate new insights into the rate-limiting barriers and mechanisms of tumor-specific targeting for nanoparticle therapeutics. Future applications of the information obtained from this project may be applied to improve the delivery and therapeutic efficacy of molecularly targeted drugs and drug combinations, which has the potential to eventually translate into novel, more effective treatment strategies. Importantly, the successful development of effective nanoparticle therapeutics for TNBC should allow us to extend these findings to the treatment of other Fn14-positive cancer types (e.g., lung, prostate, ovarian, brain).